Affinity purification of 101 residue rat cpn10 using a reversible biotinylated probe

The purification of large synthetic peptides using conventional separation techniques often results in poor yields and homogeneity due to the accumulation of chromatographically similar deletion and truncated impurities. We have developed a highly effective synthetic strategy and one-step purification procedure that is based on (i) the application of single coupling using HBTU/HOBt activation to reduce incomplete couplings, (ii) the use of N-(2-chlorobenzyloxycarbonyloxy)succinimide as a capping agent to terminate deletion sequences and (iii) the N-terminal derivatization of the complete peptidyl-resin with a reversible Fmoc-based chromatographic probe possessing enhanced physico-chemical properties (i.e. hydrophobicity, charge or affinity label). We report the application of a biotinylated probe, activated as the succinimidyl carbonate, for the purification of a 101 residue chaperonin protein from Rattus norvegicus (rat cpn10), previously synthesized using an optimized synthetic protocol. Biotinylated rat cpn10 was separated from underivatized impurities on an immobilized monomeric avidin column. Free rat cpn10 was released from avidin–agarose column with 5% aqueous triethylamine and after desalting by RP-HPLC gave 9.9% recovery. Characterization and assessment of homogeneity was achieved using ESI-MS, CZE and RP-HPLC.     

Oligonucleotide-modified screen-printed gold electrodes for enzyme-amplified sensing of nucleic acids

An electrochemical genosensor for the detection of specific sequences of DNA has been developed using disposable screen-printed gold electrodes. Screen-printed gold electrodes were firstly modified with a mixed monolayer of a 25-mer thiol-tethered DNA probe and a spacer thiol, 6-mercapto-1-hexanol (MCH). The DNA probe sequence was internal to the sequence of the 35S promoter, which sequence is inserted in the genome of GMOs regulating the transgene expression. An enzyme-amplified detection scheme, based on the coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the electroinactive alpha-naphthyl phosphate to alpha-naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry. The assay was, firstly, characterised using synthetic oligonucleotides. Relevant parameters, such as the probe concentration and the immobilisation time, the use of the MCH and different enzymatic conjugates, were investigated and optimised. The genosensor response was found to be linearly related to the target concentration between 0 and 25 nmol/L; the detection limit was 0.25 nmol/L. The analytical procedure was then applied for the detection of the 35S promoter sequence, which was amplified from the pBI121 plasmid by polymerase chain reaction (PCR). Hybridisation conditions (i.e., hybridisation buffer and hybridisation time) were further optimised. The selectivity of the assay was confirmed using biotinylated non-complementary amplicons and PCR blanks. The results showed that the genosensor enabled sensitive (detection limit: 1 nmol/L) and specific detection of GMO-related sequences, thus providing a useful tool for the screening analysis of bioengineered food samples.     

DNA sequence of the murine gamma 1 switch segment reveals novel structural elements

Immunoglobulin heavy chain switch regions are segments of DNA considered to be important in mediating class switching in B lymphocytes. Whereas these segments vary in length among the different murine isotypes, their structural organization schemes are all based on the tandem repetition of unit sequences. We previously showed that the S gamma 1 segment unexpectedly contains sequence elements that differ significantly from its prevalent unit repeat (49mer). Here we extend this preliminary characterization by determining the complete nucleotide sequence of the cloned S gamma 1 segment from BALB/c DNA. We find that S gamma 1 consists of more than 120 tandemly repeated 49mers. In addition, we show that the previously identified non-49mer sequences are part of a direct repeat element about 350 bp in length (DR II), which exists in two copies at the 5' end of S gamma 1. We also show that another unrelated direct repeat element about 500 bp long (DR I) exists near the 5' and 3' ends of S gamma 1. Thus, the structure of the S gamma 1 segment might be may be abbreviated as 5'-DRII-(49mer)15-DRI-DRII-(49mer)n-DRI , where n is between 40 and 160. Our results of Southern hybridization experiments suggest that this basic structural scheme is maintained in eight different Igh haplotypes, although S gamma 1 segments in different Igh haplotypes include different numbers of 49mer elements. Other murine S gamma segments differ in size among various Igh loci, but to a lesser extent than S gamma 1. At the level of tandemly repeated sequences, S gamma 1, S gamma 3, and S gamma 2b represent three distinct, nonoverlapping sets of sequences.     

Molecular cloning and characterization of a complete DNA copy of potato spindle tuber viroid RNA.

Double-stranded cDNA has been synthesized from RNA of a severe strain of potato spindle tuber viroid using a synthetic oligodeoxyribonucleotide as a primer. Upon cloning in bacteriophage M13mp9, two recombinant phages were selected to construct a pBR322-derived plasmid containing a complete viroid DNA copy. Elucidation of the nucleotide sequence revealed four differences with the previously established sequence of another PSTV strain, three of which were base exchanges and one a deletion.     

Insulin-like growth factor II messenger ribonucleic acids are synthesized in the choroid plexus of the rat brain

Previous studies demonstrating the presence of immunoreactive insulin-like growth factors (IGFs) and their receptors in the brain suggest a role of the IGFs in the central nervous system. IGF-II has been implicated as the predominant IGF in brain of mature animals based on studies of immunoreactive peptide and of IGF-II mRNAs. To obtain information about the sites of synthesis of IGF-II in adult rat brain, a 32P-labeled 31 base long synthetic oligodeoxyribonucleotide complementary in sequence to trailer peptide coding sequences in rat IGF-II mRNA (IGF-II 31 mer) was hybridized with coronal sections of fixed rat brain. The IGF-II 31 mer showed specific hybridization with the choroid plexus throughout rat brain, whereas in other brain regions, structures or cells, hybridization was not discernibly above background. These findings suggest that the choroid plexus is a primary site of synthesis of IGF-II, a probable source of IGF-II in cerebrospinal fluid, and a potential source of IGF-II for actions on target cells within the adult rat brain.     

Quartz crystal microbalance (QCM) affinity biosensor for genetically modified organisms (GMOs) detection

A DNA piezoelectric sensor has been developed for the detection of genetically modified organisms (GMOs). Single stranded DNA (ssDNA) probes were immobilised on the sensor surface of a quartz crystal microbalance (QCM) device and the hybridisation between the immobilised probe and the target complementary sequence in solution was monitored. The probe sequences were internal to the sequence of the 35S promoter (P) and Nos terminator (T), which are inserted sequences in the genome of GMOs regulating the transgene expression. Two different probe immobilisation procedures were applied: (a) a thiol-dextran procedure and (b) a thiol-derivatised probe and blocking thiol procedure. The system has been optimised using synthetic oligonucleotides, which were then applied to samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, certified reference materials (CRM), and real samples amplified by the polymerase chain reaction (PCR). The analytical parameters of the sensor have been investigated (sensitivity, reproducibility, lifetime etc.). The results obtained showed that both immobilisation procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples.     

Cloning of the Escherichia coli gene for the stringent starvation protein

Summary In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with ³²P at their 5-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were coloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.Abbreviations SSP stringent starvation protein - PTH phenylthiohydantoin     

Deletion pattern of the STS gene in X-linked ichthyosis in a Mexican population

BACKGROUND: X-linked ichthyosis (XLI) is an inherited disorder due to steroid sulfatase deficiency (STS). Most XLI patients (>90%) have complete deletion of the STS gene and flanking sequences. The presence of low copy number repeats (G1.3 and CRI-S232) on either side of the STS gene seems to play a role in the high frequency of these interstitial deletions. In the present study, we analyzed 80 Mexican patients with XLI and complete deletion of the STS gene. MATERIALS AND METHODS: STS activity was measured in the leukocytes using 7-[(3)H]-dehydroepiandrosterone sulfate as a substrate. Amplification of the regions telomeric-DXS89, DXS996, DXS1139, DXS1130, 5' STS, 3' STS, DXS1131, DXS1133, DXS237, DXS1132, DXF22S1, DXS278, DXS1134-centromeric was performed through PCR. RESULTS: No STS activity was detected in the XLI patients (0.00 pmoles/mg protein/h). We observed 3 different patterns of deletion. The first two groups included 25 and 32 patients, respectively, in which homologous sequences were involved. These subjects showed the 5' STS deletion at the sequence DXS1139, corresponding to the probe CRI-S232A2. The group of 32 patients presented the 3' STS rupture site at the sequence DXF22S1 (probe G1.3) and the remaining 25 patients had the 3' STS breakpoint at the sequence DXS278 (probe CRI-S232B2). The third group included 23 patients with the breakpoints at several regions on either side of the STS gene. No implication of the homologous sequences were observed in this group. CONCLUSION: These data indicate that more complex mechanisms, apart from homologous recombination, are occurring in the genesis of the breakpoints of the STS gene of XLI Mexican patients.     

The mink proopiomelanocortin gene: characterization of cDNA and chromosomal localization

A cDNA library from the mink pituitary was screened using as probe a synthetic oligodeoxyribonucleotide, 5'-TTCATGACCTCCGA-3', corresponding to the endorphin region of bovine proopiomelanocortin (POMC) cDNA. As a result, several clones containing inserts complementary to POMC mRNA were identified. The sequence of one of the fragments (585 bp, 65% of the total length of mRNA) was determined. A high degree of homology (over 80%) among the primary structures of sequences from mink, man, and bovine cDNA POMC was established. With the cloned mink cDNA fragment as probe, the DNAs from mink-Chinese hamster hybrid clones were studied. The results of segregation analysis of mink POMC sequences and mink chromosomes in the mink-Chinese hamster panel allowed us to assign the POMC gene to mink chromosome 11.     

Purification and characterization of two serine isoacceptor tRNAs from bovine mitochondria by using a hybridization assay method.

For large scale preparation of mitochondrial tRNAs, a new hybridization assay method using synthetic oligodeoxyribonucleotide probes (16-17mer) complementary to individual tRNA sequences was developed and applied for the purification of two serine isoacceptor tRNAs (tRNASerAGY and tRNASerUCN) from bovine mitochondria. It is about 100 times more sensitive than the conventional aminoacylation assay method. 2-4 A260 units each of both tRNASer isoacceptors were purified from 17.5 kg of bovine liver, and they were characterized by means of nuclease digestion, melting profiles and aminoacylation activity. It is suggested that tRNASerUCN possesses the D loop/T loop interaction like usual L-shaped tRNAs, and that tRNASerAGY lacking almost an entire D arm is aminoacylated with an efficiency not very much lower than that of tRNASerUCN.     

Precise discrimination among homologous sequences by DNA probe column chromatography.

Synthesized oligo probes were immobilized to a HPLC gel in high concentrations (30-40 O.D. per gram dry gel), packed in a column (2mm x 10cm) and incorporated into a conventional HPLC system. The system was applied to the discrimination among homologous sequences. Two probes different in sequence and length (16mer and 24mer) were investigated under isothermal and isocratic conditions. For each probe, 4 oligos of similar sequences, one perfectly matched to the probe and others containing one mismatch in the center of the chain, were synthesized. The chromatogram obtained as the results of high performance liquid affinity chromatography (HPLAC) considerably varied with the column temperature and the type of mismatches. The dependency of the deformation of elution profile upon mismatch seemed to reflect the stability of the hybrid composed of samples and immobilized probe.     

A novel probe for human DNA fingerprinting based on chi-like sequences.

A synthetic oligodeoxyribonucleotide (oligo) containing crossover initiating hotspot-like sequences was designed on the assumption that hypervariability is partly due to the presence of molecular signals which promote recombination. This oligo, when used as a probe for human DNA fingerprinting, generated individual-specific DNA band patterns. The probability of two unrelated individuals having the same DNA band pattern, using this probe, was estimated to be 1.9 x 10(-13).     

Primary structure of guinea-pig Hageman factor: sequence around the cleavage site differs from the human molecule.

The guinea-pig and human Hageman factors differ in their sensitivity to activation by particular bacterial proteinases. To understand this difference, the primary structure and cleavage site on activation of the guinea-pig molecule were determined and compared with the human molecule. By the use of a synthetic oligodeoxyribonucleotide probe which encoded a part of human Hageman factor cDNA, a cDNA clone was isolated from a lambda gt11 cDNA library of guinea-pig liver and sequenced. The cDNA clone was identified as that of guinea-pig Hageman factor by the complete identity of the deduced amino-acid sequence with the actual sequence of the amino-terminal portion of guinea-pig Hageman factor molecule and the active form. The cDNA included part of a leader sequence and the entire coding region of the Hageman factor molecule. Guinea-pig Hageman factor was composed of the same domain structures as the human counterpart with an overall 72% homology in the amino-acid sequence. However, the sequences around the cleavage site were surprisingly different; -Met351-Thr-Arg-Val-Val-Gly-Gly-Leu-Val359-(human) and -Leu338-Ser-Arg-Ile-Val-Gly-Gly-Leu-Val346-(guinea-pig). The amino-acid substitutions around the cleavage site might explain the difference in sensitivity to activation between the human and guinea-pig molecules.     

Primary structure of the beta-subunit of bovine transducin deduced from the cDNA sequence

DNA complementary to the bovine retinal mRNA coding for the beta-subunit of transducin has been cloned by screening a cDNA library with oligodeoxyribonucleotide probes. Nucleotide sequence analysis of the cloned cDNA has revealed that this polypeptide consists of 340 amino acid residues (including the initiating methionine). Furthermore, cDNA hybridizable with a transducin beta-subunit cDNA probe has been cloned from a library derived from bovine brain poly(A)+ RNA. Comparison of the cloned cDNAs, in conjunction with blot hybridization analysis and S1 nuclease mapping of poly(A)+ RNA from bovine retina, brain and liver, suggests that the mRNAs coding for the beta-subunits of transducin and other guanine nucleotide binding proteins have the same protein-coding sequence but partly different 5'-noncoding sequences.